Collagen is the most abundant protein in vertebrates, occurring in virtually every tissue, including skin, tendon, bone, blood vessel, cartilage, ligament, and teeth. Collagen serves as the fundamental structural protein for vertebrate tissues. Collagen abnormalities are associated with a wide variety of human diseases, including arthritis, rheumatism, brittle bones, atherosclerosis, cirrhosis, and eye cataracts. Collagen is also critically important in wound healing.
Collagen is a fibrous protein consisting of three polypeptide chains that fold into a triple helix (Jenkins & Raines, Nat. Prod. Rep., 19:49-59 (2002)). Mammals produce at least 46 distinct polypeptide chains that combine to form at least 28 distinct collagen types (Shoulders and Raines, Annu. Rev. Biochem. 2009. 78:929-58). In each of these variants, the polypeptide chains of collagen are composed of approximately 300 repeats of the tripeptide sequence Xaa-Yaa-Gly, where Xaa is often (but not always) a proline (Pro) residue and Yaa is often (but not always) a 4(R)-hydroxyproline (Hyp) residue, and Gly is always glycine. In connective tissue (such as bone, tendon, cartilage, ligament, skin, blood vessels, and teeth), individual collagen molecules are wound together in tight triple helices. These helices are organized into fibrils of great tensile strength (Jones & Miller, J. Mol. Biol., 218:209-219 (1991)). Varying the arrangements and cross linking of the collagen fibrils enables vertebrates to support stress in one-dimension (e.g., tendons), two-dimensions (e.g., skin), or three-dimensions (e.g., cartilage).
Collagen's biological significance has made collagen a common target for biomaterials engineering, encouraging the development of self-assembling synthetic peptide systems that mimic the triple-helical architecture of collagen. At the core of such synthetic peptide systems are collagen-mimetic peptides, or CMPs. Although many of these efforts employ non-covalent means to program strand association, the covalent cross-linking of strands remains the most robust strategy (see, e.g., Kinberger, G. A.; Cai, W. B.; Goodman, M. J. Am. Chem. Soc. 2002, 124, 15162-15163; Barth, D.; Kyrieleis, O.; Frank, S.; Renner, C.; Moroder, L. Chem.-Eur. J. 2003, 9, 3703-3714; Homg, J.-C.; Hawk, A. J.; Zhao, Q.; Benedict, E. S.; Burke, S. D.; Raines, R. T. Org. Lett. 2006, 8, 4735-4738; Khew, S. T.; Tong, Y. W. Biochemistry 2008, 47, 585-596). Indeed, cystine “knots”—complex arrangements of interstrand Cys-Cys disulfide bridges—are found in natural fibrillar and fibril-associated collagens, inspiring the use of Cys-Cys bridges in synthetic collagen-like fibrillar assemblies that extend through sticky ends (see, e.g., Koide, T.; Homma, D. L.; Asada, S.; Kitagawa, K. Bioorg. Med. Chem. Let. 2005, 15, 5230-5233; Kotch, F. W.; Raines, R. T. Proc. Natl. Acad. Sci. USA 2006, 103, 3028-3033; Yamazaki, C. M.; Asada, S.; Kitagawa, K.; Koide, T. Biopolymers 2008, 90, 816-823; Yamazaki, C. M.; Kadoya, Y.; Hozumi, Okano-Kosugi, H.; Asada, S.; Kitagawa, K.; Nomizu, M.; Koide, T. Biomaterials 2010, 31, 1925-1934).
Collagen strands associate into triple helices with a single-residue stagger that gives rise to registers with an Xaa, Yaa, and Gly residue from each strand appearing at every cross-sectional plane along the triple helix, enabling cystine residues to be installed at proximal Xaa . . . Yaa pairs (FIG. 1A). However, an examination of neighboring Xaa . . . Yaa pairs in a [(PPG)10]3 crystal structure (PDB entry 1kf6) (Berisio, R.; Vitagliano, L.; Mazzarella, L.; Zagari, A. Protein Sci. 2002, 11, 262-270) reveals the Xaa . . . Yaa Cβ . . . Cβ distance (5 Å) to be longer than the average Cβ . . . Cβ distance (4 Å) predicted for a cystine dipeptide (Ozhogina, O. A.; Bominaar, E. L. J Struct. Biol. 2009, 168, 223-233). Thus, even neighboring Xaa and Yaa positions do not allow a geometry favorable for disulfide-bond formation, and any disulfide Xaa . . . Yaa cystine bonds formed between cysteine residues on separate CMP strands would exert unfavorable strain on the triple collagen triple helix.
Consistent with this observation, natural cystine knots are known to interrupt the triple-helical structure of collagen (see Barth, D.; Kyrieleis, O.; Frank, S.; Renner, C.; Moroder, L. Chem.-Eur. J. 2003, 9, 3703-3714 Boudko, S. P.; Engel, J.; Okuyama, K.; Mizuno, K.; Bachinger, H. P.; Schumacher, M. A. J. Biol. Chem. 2008, 283, 32580-32589; Wegener, H.; Paulsen, H.; Seeger, K. J. Biol. Chem. 2014, 289, 4861-4869). However, any effect of this disruption on collagen function is compensated by the length of common native collagen strands, which have about 103 amino acid residues. In contrast, CMPs in typical synthetic assemblies are only about 30 amino acid residues long, and thus are more susceptible to the adverse impact from the strain of a interstrand cystine (cysteine-cysteine) disulfide linkage on the collagen-like triple helix.
Accordingly, there is a need in the art for compositions and methods for optimizing covalent interstrand bridges in collagen-mimetic peptides and other synthetic collagen-like biomaterials by reducing the strain associated with conventional cystine (cysteine-cysteine or Cys-Cys) disulfide linkages.